Sains Malaysiana 47(3)(2018): 499–509

http://dx.doi.org/10.17576/jsm-2018-4703-09

Transient Expression of an Immunogenic Envelope Attachment Glycoprotein of Nipah Virus in Nicotiana benthamiana

(Pengekspresan Protein Glikoprotein Virus Nipah secara Transien dalam Nicotiana benthamiana)

GAN KHAI SWAN, TAN CHON SENG, ROFINA YASMIN OTHMAN & JENNIFER

ANN HARIKRISHNA*

Centre for Research in Biotechnology for Agriculture, University of Malaya, 50603, Kuala Lumpur, Malaysia

Received: 23 March 2017/Accepted: 13 October 2017

ABSTRACT

The Nipah virus is highly virulent to swine and humans. The envelope attachment glycoprotein (G) of Nipah virus plays a key role in viral entry and induction of neutralizing antibody in mammalian hosts, thus is considered a good candidate for vaccine development. Plant transient expression systems are gaining recognition as a viable alternative for the production of vaccine antigens. In this study, we expressed the Nipah virus G protein heterologously in Nicotiana benthamiana using an agroinfiltration approach. The highest expression of recombinant G protein in N. benthamiana at RNA and protein levels was detected on day 9 post-infiltration. Western blot analysis demonstrated that the purified G protein reacted specifically with rabbit anti-Nipah Virus serum, indicating its potential for vaccine use.

Keywords: Biotechnology; KDEL; plants; tobacco PR1a; vaccine

ABSTRAK

Virus Nipah adalah virus yang sangat virulen bagi ternakan khinzir dan manusia. Pelekat sampul sel gliko protein G virus Nipah memainkan peranan penting dalam kemasukan virus dan peneutralan antibodi di dalam sel perumah mamalia. Oleh itu, ia dianggap sebagai satu calon yang baik untuk dijadikan vaksin. Memandangkan sistem asai transien dalam tumbuhan semakin diberi perhatian sebagai cara alternatif penghasilan vaksin, protein G virus Nipah telah diekspres dalam tumbuhan Nicotiana benthamiana menerusi kaedah agroinfiltrasi di dalam kajian ini. Dalam kajian ini, pengekspresan RNA dan protein G dalam Nicotiana benthamiana mencapai tahap maksimum pada hari ke-9 selepas infiltrasi. Protein G yang ditulenkan menunjukkan tindak balas yang khusus dengan serum arnab anti-Nipah virus dalam analisis pedapan Western. Penemuan ini menunjukkan protein rekombinan G virus Nipah berpotensi dijadikan vaksin.

Kata kunci: Bioteknologi; KDEL; tembakau PR1-a; tumbuhan; vaksin

REFERENCES

Agrawal, G.K., Jwa, N.S. & Rakwal, R. 2000. A novel rice (Oryza sativa L.) acidic PR1 gene highly responsive to cut, phytohormones, and protein phosphatase inhibitors. Biochem. Biochemical and Biophysical Research Communications 274(1): 157-165.

Bossart, K.N., Crameri, G., Dimitrov, A.S., Mungall, B.A., Feng, Y.R. & Patch, J.R., Choudhary, A., Wang, L.F., Eaton, B.T. & Broder, C.C. 2005. Receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble g glycoprotein of hendra virus. Journal of Virology 79(11): 6690-6702.

Broder, C.C. 2012 Henipavirus outbreaks to antivirals: The current status of potential therapeutics. Curr. Opin. Virol. 2: 176-187.

Broder, C.C., Weir, D.L. & Reid, P.A. 2016. Hendra virus and Nipah virus animal vaccines. Vaccine 34(30): 3525-3534.

Chen, Q. & Lai, H. 2015. Gene delivery into plant cells for recombinant protein production. BioMed. Research International 10: 1-10.

Chua, K.B., Bellini, W.J., Rota, Harcourt B.H., Tamin, A., Lam, S.K, Ksiazek, T.G., Rollin, P.E., Zaki, S.R., Shieh, W., Goldsmith, C.S., Gubler, D.J., Roehrig, J.T., Eaton, B., Gould, A.R., Olson, J., Field, H., Daniels, P., Ling, A.E., Peters, C.J., Anderson, L.J. & Mahy, B.W. 2000. Nipah virus: A recently emergent deadly paramyxovirus. Science 288(5470): 1432-1435.

Chua, K.B., Goh, K.J., Wong K.T., Kamarulzaman, A., Tan, P.S., Ksiazek, T.G., Zaki, S.R., Paul, G., Lam, S.K. & Tan, C.T. 1999. Fatal encephalitis due to Nipah virus among pig-farmer in Malaysia. The Lancet 354(9186): 1257-1259.

Clayton, B.A., Middleton, D., Arkinstall, R., Frazer L., Wang, L.F. & Marsh, G.A. 2016. The nature of exposure drives transmission of Nipah viruses from Malaysia and Bangladesh in ferrets. PLoS Negl. Trop. Dis. 10: e0004775.

Daniell, H., Lee, S.B., Panchal, T. & Wiebe, P.O. 2001. Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J. Mol. Biol 311(5): 1001-1009.

DeBuysscher, B.L., Scott, D., Marzi, A., Prescott, J. & Feldmann, H. 2014. Single-dose live-attenuated Nipah virus vaccines confer complete protection by eliciting antibodies directed against surface glycoproteins. Vaccine 32(22): 2637-2644.

DeBuysscher, B.L., Scott, D., Thomas, T., Feldmann, H. & Prescott, J. 2016. Peri-exposure protection against Nipah virus disease using a single-dose recombinant vesicular stomatitis virus-based vaccine. Vaccines 1: 16002.

Eaton, B.T., Broder, C.C., Middleton, D. & Wang, L.F. 2006. Hendra and Nipah viruses: Different and dangerous. Nature Rev. Microbiol. 4(1): 23-35.

Eshaghi, M., Tan, W.S., Chin, W.K. & Yusoff, K. 2005. Purification of the extra-cellular domain of nipah virus glycoprotein produced in Escherichia coli and possible application in diagnosis. Journal of Biotechnology 116(3): 221-226.

Faye, L., Boulaflous, A., Benchabane, M., Gomord, V. & Michaud, D. 2005. Protein modifications in the plant secretory pathway: Current status and practical implications in molecular pharming. Vaccine 23(15): 1770-1778.

Fischer, R., Stoger, E., Schillberg, S., Christou, P. & Twyman, R.M. 2004. Plant-based production of biopharmaceuticals. Current Opinion in Plant Biology 7(2): 152-158.

Gan, G.S., Tan, C.S., Othman, R.Y. & Harikrishna, J.A. 2015. Glucose and low temperature enhanced plasmid stability for increasing expression of Nipah virus glycoprotein in Escherichia coli. Research Journal of Biotechnology 10(9): 1-10.

Geisbert, T.W., Feldmann, H. & Broder, C.C. 2012. Animal challenge models of Henipavirus infection and pathogenesis. Henipavirus 10: 153-177.

Gomord, V., Sourroulle, C., Fitchette, A.C., Bardor, M., Pagny, S. & Lerouge, P. & Faye, L. 2004. Production and glycosylation of plant-made pharmaceuticals: The antibodies as a challenge. Plant Biotechnology Journal 2(1): 83-100.

Halpin, K., Hyatt, A.D., Fogarty, R., Middleton, D., Bingham, J., Epstein, J.H., Rahman, S.A, Hughes, T., Smith, C., Field, H.E. & Daszak, P. 2011. Pteropid bats are confirmed as the reservoir hosts of henipaviruses: A comprehensive experimental study of virus transmission. The American Journal of Tropical Medicine and Hygiene 85(5): 946-951

Harcourt, B.H., Tamin, A., Ksiazek, T.G., Rollin P.E., Anderson L.J. & Bellini W.J. & Rota, P.A. 2000. Molecular characterization of Nipah virus, a newly emergent paramyxovirus. Virology 271(2): 334-349.

Herriman, R. 2015 Bangladesh reports nine Nipah virus cases to date in 2015. Outbreak News Today. Retrieved from http:// outbreaknewstoday.com/bangladesh-reports-nine-nipah-virus-cases-to-date-in-2015/.

Islam, M.S., Sazzad, H.M., Satter, S.M., Sultana, S., Hossain, M.J. & Hasan, M., Islam, M.S., Sazzad, H.M., Satter, S.M., Sultana, S., Hossain, M.J., Hasan, M., Rahman, M., Campbell, S., Cannon, D.L., Ströher, U. & Daszak, P. 2016. Nipah virus transmission from bats to humans associated with drinking traditional liquor made from date palm sap, Bangladesh, 2011-2014. Emerging Infectious Diseases 22(4): 664-670.

Johansen, L.K. & Carrington, J.C. 2011. Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiology 126(3): 930-938.

Kalthoff, D., Giritch, A., Geisler, K., Bettmann, U. & Klimyuk, V. 2010. Immunization with plant-expressed hemagglutinin protects chickens from lethal highly pathogenic avian influenza virus H5N1 challenge infection. Journal of Virology 84(22): 12002-12010.

Kanagarajan, S., Tolf, C., Lundgren, A., Waldenstrom, J. & Brodelius, P.E. 2013. Transient expression of Hemagglutinin antigen from low pathogenic avian influenza A (H7N7) in Nicotiana benthamina. Plos One 7: e33010.

Kapila, J., De Rycke, R., Van, Montagu, M. & Angenon, G. 1997. An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Science 122(1): 101-108.

Lindbo, J.A. 2007. TRBO: A high-efficiency Tobacco mosaic virus RNA-based overexpression vector. Plant Physiology 145(4): 1232-1240.

Liu, Q., Bradel-Tretheway, B., Monreal, A.I., Saludes, J.P., Lu, X., Nicola, X.V. & Aguilar, H.C. 2015. Nipah virus attachment Glycoprotein stalk C-terminal region links receptor binding to fusion triggering. Journal of Virology 80(3): 1839-1850.

Liu, Y.J., Xiao, C., Wang, G.P., Xuan, H. & Tu, C.C. 2007. Expression of Nipah virus structural proteins F1 and G and preparation of hyperimmune antisera against two proteins. Acta Microbiologica Sinica 47(3): 465-470.

Lo, M.K., Bird, B.H., Chattopadhyay, A., Drew, C.P., Martin, B.A., Coleman J.D. Rose, J.K., Nichol, S.T. & Spiropoulou, C.F. 2014. Single-dose replication-defective VSV-based Nipah virus vaccines provide protection from lethal challenge in Syrian hamsters. Antiviral Research 101: 26-29.

Lomonosoff, G.P. 2015. Rapid high-yield expression of a candidate influenza vaccine based on the ectodomain of M2 protein linked to flagellin in plants using viral vectors. BMC Biotechnology 15(1): 42.  

Luby, S.P., Gurley, E.S. & Hossain, M.J. 2012. Transmission of Human Infection with Nipah Virus. In Improving Food Safety Through a One Health Approach, Workshop Summary. Institute of Medicine (US): National Academies Press. p. A11.

Luby, S.P., Hossain, M.J., Gurley, E.S., Ahmed, B.N., Banu, S., Khan, S.U., Homaira, N., Rota, P.A., Rollin, P.E., Comer, J.A., Kenah, E., Ksiazek, T.G. & Rahman, M. 2009. Recurrent zoonotic transmission of Nipah virus into humans, Bangladesh, 2001-2007. Emerging Infectious Diseases 15(8): 1229-1235.

Ma, S., Huang, Y., Davis, A., Yin, Z., Mi, Q., Menassa, R., Brandle, E. & Jevnikar, A.M. 2005. Production of biologically active human interleukin-4 in trasngenic tobacco and potato. Plant Biotechnology Journal 3(3): 309-318.

Mason, H.S., Lam, D.M. & Arntzen, C.J. 1992. Expression of hepatitis B surface antigen in transgenic plants. Proceedings of the National Academy of Sciences 89(24): 11745-11749.

Mayo, M.A. 2002. A summary of taxonomic changes recently approved by ICTV. Archives of Virology 147(8): 1655-1656.

McEachern, J.A., Bingham, J., Crameri, G., Green, D.J., Hancock, T.J., Middleton D., Feng, Y.R., Broder, C.C., Wang, L.F. & Bossart, K.N. 2008. A recombinant subunit vaccine formulation protects against lethal Nipah virus challenge in cats. Vaccine 26(31): 3842-3852.

Mett, V., Musiychuk, K., Bi, H., Farrance, C.E. & Horsey, A. 2008. A plant-produced influenza subunit vaccine protects ferrets against virus challenge. Influenza and Other Respirotory Viruses 2(1): 33-40.

Mire, C.E., Versteeg, K.M., Cross, R.W., Agans, K.N., Fenton, K.A., Whitt, M.A. & Geisbert, T.W. 2013. Single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal Nipah virus disease. Virology Journal 10(1): 353.

Musiychuk, K., Stephenson, N., Bi, H., Farrance, C.E., Orozovic, G., Brodelius, M., Brodelius, P., Horsey, A., Ugulava, N., Shamloul, A.M., Mett, V., Rabindran, S., Streatfield, S.J. & Yusibov, V. 2007. A launch vector for the production of vaccine antigens in plants. Influenza and Other Respiratory Viruses 1(1): 19-25.

Nochi, T., Takagi, H., Yuki, Y., Yang, L., Masumura, T., Mejima, M., Nakanishi, U., Matsumura, A., Uozumi, A., Hiroi, T., Morita, S., Tanaka, K., Takaiwa, F. & Kiyono, H. 2007. Rice-based mucosal vaccine as a global strategy for cold-chain-and needle-free vaccination. Proceedings of the National Academy of Sciences 104(26): 10986-10991.

Nuttall, J., Vine, N., Hadlington, J.L., Drake, P., Frigerio, L. & Ma, J.K. 2002. ER-resident chaperone interactions with recombinant antibodies in transgenic plants. The FEBS Journal 269(24): 6042-6051.

Pallister, J.A., Klein, R., Arkinstall, R., Haining, J., Long, F., White, J.R., Payne, J., Feng, Y.R., Wang, L.F., Broder, C.C. & Middleton, D. 2013. Vaccination of ferrets with a recombinant G-glycoprotein subunit vaccine provides protection against nipah virus disease for over 12 months. Virology Journal 10(1): 237.  

Parashar, U.D., Sunn, L.M., Ong, F., Mounts, A.W., Arif, W.T., Ksiazek, T.G., Kamaluddin, M.A., Mustafa, A.N., Kaur, H., Ding, L.M., Othman, G., Radzi, H.M., Kitsutani, P.T., Stockton, P.C., Arokiasamy, J., Gary, H.E.J. & Anderson, L.J. 2000. Case-control study of risk factors for human infection with a novel zoonotic paramyxovirus, Nipah virus, during a 1998–1999 outbreak of severe encephalitis in Malaysia. The Journal of Infectious Diseases 181(5): 1755-1759.

Paton, N.I., Leo, Y.S., Zaki, S.R., Auchus, A.P., Lee, K.E., Ling, A.E., Chew, S.K, Ang, B., Rollin, P.E., Umapathi, T., Sng, I., Lee, C.C., Lim, E. & Ksiazek, T.G. 1999. Outbreak of Nipah virus infection among abattoir workers in Singapore. The Lancet 354(9186): 1253-1257.

Peng, R.H., Yao, Q.H., Xiong, A.S., Cheng, Z.M. & Li, Y. 2006. Codon-modifications and an endosplasmic recticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola. Plant Cell Reports 25(2): 124-132.

Petruccelli, S., Otegui, M.S., Lareu, F., Tran Dihn O., Fitchette, A.C., Circosta, A., Rumbo, M., Bardor, M., Carcamo, R., Gomord, V. & Beachy, R.N. 2006. A KDEL-tagged monoclonal antobody is efficiently retained in the endoplasmic reticulum in leaves but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnology Journal 4(5): 511-527.

Petukhova, N.V., Gasanova, T.V., Stepanova, L.A., Rusova, O.A., Patopchuk M.V., Korotkov, A.V. Skurat, E.V,, Tsybalova, L.M., Kiselev, O.I., Ivanov, P.A. & Atabekov, J.G. 2013. Immunogenicity and protection efficacy of candidate universal influenza Ananovaccine produced in plants by Tabacco mosaic virus-based vectors. Current Pharmaceutical Design. 19(31): 5587-5600.

Pogrebnyak, N., Golovkin, M., Andrianov, V., Spitsin, S., Smirnov, Y., Egolf, E. & Koprowski, H. 2005. Severe acute respiratory syndrome (SARS) S protein production in plants: Development of recombinant vaccine. Proceedings of the National Academy of the United States of America 102(25): 9062-9067.

Prescott, J., DeBuysscher, B.L., Feldmann, F., Gardner, D.J., Haddock, E., Martellaro, C., Scott. D. & Feldmann, H. 2015. Single-dose live-attenuated vesicular stomatitis virus-based vaccine protects African green monkeys from Nipah virus disease. Vaccine 33(24): 2823-2829.

Pua, T.L., Loh, H.S., Massawe, F., Tan, C.S. & Omar, A.R. 2012. Expression of insoluble influenza neuraminidase Type 1(NA1) protein in tobacco. Journal of Tropical Life Science 2(3): 62-71.

Qiu, X., Wong, G., Audet, J., Bello, A., Femando, L., Alimonti, J.B., Fausther-Bovendo, H., Wei, H., Aviles, J., Hiatt, E., Johnson, A., Morton, J., Swope, K., Bohorov, O., Bohorova, N., Goodman, C., Kim, D., Pauly, M.H., Velasco, J., Pettitt, J., Olinger, G.G., Whaley, K., Xu, B., Strong, J.E. & Zeitlin, L. 2014. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature 514(7520): 47-53.

Roy, G., Weisburg, S., Rabindran, S. & Yusibov, V. 2010. A novel two-component tobacco mosaic virus-based vector system for high-level expression of multiple therapeutic proteins including a human monoclonal antibody in plants. Virology 405(1): 93-99.

Rybicki, E.P. 2014. Plant-based vaccines against viruses. Virology Journal 11(1): 205.

Sainsbury, F., Liu, L. & Lomonossoff, G.P. 2009b. Cowpea mosaic virus-based systems for the expression of antigens and antibodies in plants. Recombinant Proteins From Plants: Methods and Protocols 483: 25-39.

Sainsbury, F. & Lomonossoff, G.P. 2008. Extremely high-evel and rapid transient protein production in plants without the use of viral replication. Plant Physiology 148(3): 1212-1218.

Sainsbury, F., Thuenemann, E.C. & Lomonossoff G.P. 2009a. pEAQ: Versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Journal Plant Biotechnology 7(7): 682-693.

Satterfield, B.A., Dawes, B.A. & Milligan, G.N. 2016. Status of vaccine research and development of vaccines for Nipah virus. Vaccine 34(26): 2971-2975.

Scholthof, H.B., Morris, T.J. & Jackson, A.O. 1993. The capsid protein gene of tomato bushy stunt virus is dispensable for systemic movement and can be replaced for localized expression of foreign genes. Molecular Plant-Microbe. Interactions Journal 6(3): 309-322.

Schouten, A., Roosien, J., Van Engelen, F.A., de Jong, G.A., Borst-Vrenssen, A.W., Zilverentant, J.F., Bosch, D., Stiekema, W.J., Gommers, F.J., Schots, A. & Bakker, J. 1996. The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. Plant Molecular Biology 30(4): 781-791.

Sheludko, Y.V., Sindarovska, Y.R., Gerasymenko, I.M., Bannikova, M.A. & Kuchuk, N.V. 2007. Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression. Biotechnology Bioengeneering 96(3): 608-614.

Shoji, Y., Chichester, J.A., Bi H., Musiychuk, K. & de la Rosa, P. 2008. Plant-expressed HA as a seasonal influenza vaccine candidate. Vaccine 26: 2930-2934.

Shoji, Y., Chichester, J.A., Jones, M., Manceva, S.D. & Damon, E. 2011. Plant-based rapid production of recombinant subunit hemagglutinin vaccines targeting H1N1 and H5N1 influenza. Hum. Vaccines 7(sup1): 41-50.

Stoger, E., Sack, M., Perrin, Y., Vaquero, C., Torres, E., Twyman, R.M., Christou, P. & Fisher, R. 2002. Practical considerations for pharmaceutical antibody production in different crop systems. Molecular Breeding 9(3): 149-158.

Sun, Q.Y., Ding, L.W., Lomonossoff, G.P., Sun Y.B., Luo M., Li, C.Q. Jiang, L. & Xu, Z.F. 2011. Improved expression and purification of recombinant human serum albumin from transgenic tobacco suspension culture. Journal of Biotechnology 155(2): 164-172.

Voinnet, O., Rivas, S., Mestre, P. & Baulcombe, D. 2003. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. The Plant Journal 33(5): 949-956.

Wang, L.F., Yu, M., Hansson, E., Pritchard, L.I., Shiell, B., Michalski, W.P. & Eaton, B.P. 2000. The exceptionally large genome of Hendra virus: Support for creation of a new genus within the family Paramyxoviridae. Journal of Virology 74(21): 9972-9979.

Weigel, D. & Glazebrook, J. 2006. Transformation of agrobacterium using the freze-thaw method. Cold Spring Harbor Protocols doi: 10.1101/pdb.prot4666.

Weingart, H.M., Berhane, Y., Caswell, J.L., Loosmore, S., Audonnet J.C., Roth, J.A. & Czub, M. 2006. Recombinant Nipah virus vaccines protect pigs against challenge. Journal of Virology 80(16): 7929-7938.

Wigdorovitz, A., Carrillo, C., Samtos, M., Trono, K. & Peralta, A. 1999. Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology 255(2): 347-353.

Wong, K.T. & Ong, K.C. 2011. Pathology of acute henipavirus infection in humans and animals. Patholog. Res. Int. 2011: Article ID. 567248.

Wroblewski, T., Tomczak, A. & Michelmore R. 2005. Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnology Journal 3(2): 259-273.

Wydro, M., Kozubek, E. & Lehmann, P. 2006. Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana. Acta. Biochimica Polonica 53(2): 289-298.

Yang, J., Barr, L.A., Fahnestock, S.R. & Liu, Z.B. 2005. High yield recombinant silk-like protein production in transgenic plants through protein targeting. Transgenic Research 14(3): 313-324.

Yoneda, M., Georges-Courbot, M.C., Ikeda, F., Ishii, M. & Nagata, N. 2013. Recombinant Measles virus vaccine expressing the Nipah virus glycoprotein protects against lethal Nipah virus challenge. PloS ONE 8: e58414.

Yusibov, V., Hooper, D.G., Spitsin, S., Fleysh, N. & Kean, R.B. 2002. Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine. Vaccine 20: 3155-3164.

Zhang, C., Bradshaw, J.D., Whitham, S.A. & Hill, J.H. 2010. The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing. Plant Physiology 153(1): 52-65.

*Corresponding author; email: jennihari@um.edu.my